Method of culturing liver cells over long time

ABSTRACT

A method of culturing hepatocytes for a long term, characterized in that fresh hepatocytes are maintained in a medium at 15 to 30° C. for 1 to 6 days, followed by culturing under physiological conditions. The method of the present invention enables hepatocytes to be cultured for a long term without inviting reduction in enzymatic activity or enzyme-inducing activity. Moreover, since the cells can be maintained at around room temperature for 1 to 6 days, the method is very useful for long-term transportation of hepatocytes after isolation.

TECHNICAL FIELD

The present invention relates to a method of culturing fresh hepatocytesover a long period of time while maintaining enzymatic activities andenzyme-inducing activities of the cells.

BACKGROUND ART

Hepatocytes produce not only albumin, but also drug metabolizing enzymessuch as those belonging to the cytochrome P450 (CYP) family, andcoagulation-fibrinolysis-related enzymes such as α1-antitrypsin.Therefore, culture products of fresh hepatocytes are widely used formeasuring the activities of such enzymes.

Typically, hepatocytes that are collected from the liver are stored in acool place at 4° C. or lower so as not to permit deactivation ofenzymes, and thereafter, the temperature is returned to about 37° C. andthe cells are cultured for a long period of time before they are usedfor performing a variety of tests.

In recent years, as human hepatocytes have become difficult to obtain, aneed arises for acquired hepatocytes to be stored for a long period oftime or transported a long distance (e.g., cross-border transportation).To meet such need, fresh hepatocytes must be stored over at least 2 dayswithout inviting any loss of enzymatic activities or enzyme-inducingactivities.

However, in conventional low-temperature storage at not higher than 4°C., enzyme activities and enzyme-inducing activities are generallylowered by 30 to 60% during storage of 2 or more days, thus hamperingcorrect measurement of such activities.

Accordingly, an object of the present invention is to provide a methodof culturing hepatocytes over a long period of time without permittingreduction in enzymatic activities or enzyme-inducing activities of thecells.

DISCLOSURE OF THE INVENTION

The present inventors have studied conditions under which freshhepatocytes can be satisfactorily cultured for a long term, and haveobtained the following findings. That is, when hepatocytes are stored atabout at 37° C., which represents a physiological condition, enzymaticactivities and enzyme-inducing activities of the cells are maintained atcertain appreciable levels as compared with the case where the cells arestored at a conventionally employed low temperature (4° C.). However,when the cells are stored at room temperature; specifically between 15and 30° C., for 1 to 6 days, and then returned to physiologicalconditions and cultured, quite unexpectedly, even higher enzymaticactivities and enzyme-inducing activities are retained as compared withthe case of the storage at 37° C., and the activities remain persistentfor long periods of time, and when the hepatocytes are transported along distance, biological tests such as drug metabolism enzyme-inducingtests are performed accurately. The present invention has beenaccomplished on the basis of these findings.

Accordingly, the present invention provides a method of culturinghepatocytes for a long term, characterized in that fresh hepatocytes aremaintained in a medium at 15 to 30° C. for 1 to 6 days, followed byculturing under physiological conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the urea synthesis (i.e., ureagenesis) capacity of monkeyhepatocytes which were cultured after storage for 48 hours under avariety of conditions.

FIG. 2 shows the α1-antitrypsin synthesis capacity of monkey hepatocyteswhich were cultured after storage at 25° C. for 48 hours.

FIG. 3 shows the α1-antitrypsin synthesis capacity of monkey hepatocyteswhich were cultured after storage at 25° C. for 96 hours.

FIG. 4 shows the CYP3A4 inducing capacity of monkey hepatocytes whichwere cultured after storage at 25° C. for 48 hours (RIF representsexposure to rifampicin, whereas UT represents non-exposure torifampicin).

FIG. 5 shows the urea synthesis (i.e., ureagenesis) capacity of humanhepatocytes which were cultured after storage at 25° C. for 48 hours.

FIG. 6 shows the urea synthesis capacity of human hepatocytes which werecultured after storage at 25° C. for 96 hours.

FIG. 7 shows the urea synthesis capacity of human hepatocytes which werecultured after storage at 25° C. for 144 hours.

FIG. 8 shows the albumin synthesis capacity of human hepatocytes whichwere cultured after storage at 25° C. for 96 hours.

FIG. 9 shows the α1-antitrypsin synthesis capacity of human hepatocyteswhich were cultured after storage at 25° C. for 96 hours.

FIG. 10 shows the CYP3A4 inducing capacity of human hepatocytes whichwere cultured after storage at 25° C. for 48 hours, 96 hours, or 144hours (RIF represents exposure to rifampicin, whereas UT representsnon-exposure to rifampicin).

BEST MODES FOR CARRYING OUT THE INVENTION

The hepatocytes to which the method of the present invention is appliedare fresh hepatocytes which have not been subjected to subculturingafter removal from the organism. In other words, they are hepatocytes ofprimary culture. Examples of the hepatocytes include those from amammal, in particular, those from the primates. Human hepatocytes arevery much preferred.

According to the present invention, fresh hepatocytes must be maintainedin a medium at 15 to 30° C. for 1 to 6 days. When the hepatocytes aremaintained at a temperature below 15° C., for example, at 4° C. (whichis the conventional storage temperature), the enzymatic activities andenzyme-inducing activities of the hepatocytes decrease. Likewise, whenthe hepatocytes are maintained at a temperature higher than 30° C., forexample, at about 37° C. (which is a physiological condition), theenzymatic activities and enzyme-inducing activities of the hepatocytesdecrease even further as compared with the case where the cells arestored at 15 to 30° C. The temperature at which the hepatocytes aremaintained is preferably 18 to 27° C., more preferably 20 to 25° C.

The medium to be used in the present invention for maintaining freshhepatocytes may be a conventional one used for animal cells. Examples ofsuch a medium include Lanford's medium, William's E (WME) medium, Isom'smedium, Leibovitz L15 medium, polyethylene-glycol-added Leibovitz L15medium, and any combinations thereof. A particularly preferred mediumcontains at least Lanford's medium.

The number of the hepatocytes added to a medium for maintaining freshhepatocytes is 0.5×10⁶ to 10×10⁶ cell/mL, preferably 1×10⁶ to 10×10⁶cell/mL.

The period of time during which the hepatocytes are maintained at 15 to30° C. is 1 to 6 days, preferably 2 to 6 days.

After the hepatocytes are maintained at 15 to 30° C. for 1 to 6 days,the cells are cultured under physiological conditions. Preferably, sucha culture under physiological conditions is performed in a conventionalmedium at 37±1° C. As used herein, no particular limitation is imposedon the medium so long as it can be used for culturing conventionallyemployed animal cells. Preferably, the medium is the same as thatemployed for maintaining the aforementioned fresh hepatocytes. Inparticular, use of a medium containing at least the Lanford's medium ispreferred.

When hepatocytes are subjected to continuous culturing underphysiological conditions as described above, the enzymatic activitiesand enzyme-inducing activities of the cells do not drop over a prolongedperiod; i.e., over one month or more, and therefore, the hepatocytes canbe used for accurate measurement of enzymatic activities or drugmetabolizing enzyme-inducing activities. Examples of measurableenzymatic activities include α1-antitrypsin activity,glutamine-S-transferase (GST), GOT, GPT, ureagenesis, and albuminsynthesis capacity. Example of enzyme-inducing activities includes thosefor inducing enzymes belonging to the CYP family, such as CYP1A1,CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E6, and CYP3A4.

Procurement of hepatocytes, in particular human hepatocytes, is noteasy. Particularly when the site where hepatocytes are obtained from anorganism is remote from the sites where tests are to be carried out, asin the case of cross-border situation, transportation of hepatocyteswould typically need 2 or more days. The method of the present inventionis very suitable in cases where tests are performed after long-distancetransportation of the cells.

EXAMPLES

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

A. Materials and Methods

(1) Primary Culture of Hepatocytes

Human livers were removed from four patients. Monkey livers wereobtained from young male monkeys.

Human hepatocytes were collected from the liver that had been subjectedto lobectomy through a known method (Drug Metab. Dispos. 18: 595-606(1990), Mol. Pharmacol. 41: 1047-1055 (1992), J. Pharmacol. Exp. Ther.269: 384-392 (1994)), and cultured. Viability of the cells to be platedwas checked by the trypan blue exclusion test, and was found to be 80 to90%. The hepatocytes were seeded in Isom's medium placed in a cultureflask (25 cm²) coated with collagen I (5 μg/cm²) so as to attainconfluency (12.4×10⁴ cells/cm²). The medium employed was a 1:1 mixtureof Ham F12 and William's E, which was prepared as described in Proc.Nat. Acad. Sci. USA; 81: 6378-6382 (1984). During the first 4 hoursafter the cells were seeded, 5% calf serum was added to thereby promoteadhesion of cells. Four hours after plating of hepatocytes, thecalf-serum-added standard medium was removed, and instead, Lanford'smedium was added. Thus, on day 1 of culture, the medium was exchanged,and subsequently, the culture product of hepatocytes were maintainedunder humid conditions of 5% CO₂ at 37° C. for 3 or 4 days. In thismanner, once a hepatocyte culture system was established, the Lanford'smedium was removed through aspiration. The interior of the flask waspurged with 5%-CO₂-equilibrated 95% air, and filled with the Lanford'smedium (abbreviated as “Lnf”), 5% polyethylene glycol-added LeibovitzL15 (Sigma Chemical Co.) medium (abbreviated as “L15+5% PEG”), or Isom'smedium (37° C.). The flask was then maintained for 2, 4 or 6 days underice-cold conditions or at room temperature. Subsequently, the medium ofthe hepatocyte-containing flask was again changed to Lnf at 37° C. forlong-term culturing. In order to assess the effects of medium,temperature, and storage period on the viability and functions ofhepatocytes, the cells collected 24 hours after every medium exchangewere tested for several parameters representing the phenotypes of thecells. Specifically, the tested parameters included urea synthesiscapacity (through enzymatic/spectrometric analysis), and α1-antitrypsin-or albumin-synthesizing capacity (through western blotting). The resultsare shown in a comparative manner with the initial value and the data ofthe control group (control: a group which was maintained at 37° C. inLnf during the test period). Moreover, response of cells in terms ofinduction of cytochrome P450 enzyme (induction of CYP3A4 by rifampicin)was also studied. This response is considered an index which best tellswhether or not liver-specific phenotypes are maintained, and therefore,serves as a critical factor in development of drugs, as it can predictdrug interaction.

(2) Medium Composition

The medium compositions employed are shown in Tables 1, 2, and 3. TABLE1 Lanford's medium (Lnf) 0.5 mg/mL BSA 1 μM Hydrocortisone 10 μg/mLInsulin 50 ng/mL Epidermal growth factor (EGF) 2 μg/mL Glucagon 5 μg/mLLinolenic acid 0.1 μM Selenium acetate 2 ng/mL Cholera toxin 20 ng/mLHepatocyte growth factor 5 μg/mL Transferrin 1 μM Ethanolamine 100 ng/mLProlactin 100 U/mL Penicillin 100 μg/mL Streptomycin 25 μg/mL Fungizone

TABLE 2 L15 + 5% PEG Leibovitz L15 (product of Sigma Chemical Co.) 5%Polyethylene glycol Leibovitz L15 (mg/L) Inorganic salt CaCl₂ 140 KCl400 KH₂PO₄ 60 MgCl₂ 94 MgSO₄ 8000 NaCl 190 Na₂HPO₄ Other ingredientsD(+)Galactose 900 Phenol red 10 Sodium pyruvate 550 Amino acid L-Alanine225 L-Arginine 500 L-Asparagine 250 L-Cysteine 120 L-Glutamine 300Glycine 200 L-Histidine 250 L-Isoleucine 250 L-Leucine 125 L-Lysine 75L-Methionine 75 L-Phenylalanine 125 L-Serine 200 L-Threonine 300 L-Tryptophan 20 L-Tyrosine 300 L-Valine 100 Vitamins CalciumD-pantothenate 1 Choline chloride 1 Folic acid 1 Inositol 2 Niacinamide1 Pyridoxine HCl 1 Riboflavin-5-phosphate 2H₂O 0.1 Thiaminemonophosphate 1

TABLE 3 Isom's medium 2.4 mM Glutamine 0.1 μM Dexamethasone (DEX) 2μg/mL Insulin 50 μg/mL Transferrin 3.6 μM Linolenic acid 66.8 μMEthanolamine 0.4 mM Sodium pyruvate 250 μM Ascorbic acid 100 U/mLPenicillin 100 μg/mL Streptomycin 25 μg/mL Fungizone 0.5 μM Fluconazole(3) Preparation of Lysate

From the cultured cells, lysate was prepared through a known method byuse of a centrifuge, and stored until use. The protein concentration ofthe lysate was measured through the bicinchoninate method (PierceChemical Company).

(4) Quantification of Serum Protein Contained in an ExtracellularCulture Medium with Which the Hepatocytes are Cultured

Quantification of albumin and α1-antitrypsin contained in anextracellular culture medium (5 to 20 μL medium) was performed throughwestern blotting employing a human-protein-specific antibody. Mediumsamples were collected from 2 flasks (in which the cells were cultured).The collected samples were combined, followed by centrifuging at11,000×g for 5 minutes and storing at −80° C. until use for analysis.

(5) Quantification of CYPs Contained in Cultured Hepatocytes

In accordance with the method described in Hepatology 22: 1143-1153(1995), CYPs were quantified by use of a specific polyclonal ormonoclonal antibody through immunoblotting of a lysate. As a standard tobe applied to each gel, a microsome containing genetically expressedCYP2C9, CYP2C19, or CYP3A4 (Gentest) was employed.

The lysate was subjected to electrophoresis (SDS 10% polyacrylamide gel)before transferring onto nitrocellulose membrane. The blots were allowedto develop by use of an ECL (Enhanced ChemiLuminescence) (Amersham). Therelative amount of CYP was calculated through scanning by means of an LEprogram, and quantitative analysis by means of an NIH image 1.6/ppcprogram.

(6) Urea Synthesis Capacity of the Cultured Hepatocytes Contained in anExtracellular Culture Medium

The urea synthesis capacity was measured by an enzymatic/spectrometricassay kit (Sigma Chemical Co.).

B. Results

(1) Effects of Storage Temperature on Urea Synthesis Capacity

Monkey hepatocytes were maintained in Lnf or L-15+5% PEG for 2 days at0° C. or 25° C. Subsequently, the cells were cultured at 37° C. in Lnf.The urea synthesis capacities of the monkey hepatocytes after culturingfor 1, 2, 3, 8, or 11 days are shown in FIG. 1. As is clear from FIG. 1,as compared with the urea synthesis capacity of the cells of the controlgroup which were cultured after having been stored at 37° C. in Lnf, theurea synthesis capacities of the cell groups which were cultured afterhaving been stored at 0° C. were found to be lowered in both of thefollowing cases; i.e., the case where the cells were cultured in Lnf andthe case where the cells were cultured in L-15+5% PEG. In contrast, thecell groups which were cultured in Lnf following storage at 25° C. andthe cell groups which were cultured in L-15+5% PEG following storage at25° C. were both found to maintain urea synthesis capacities higher thanthose of corresponding control groups. Lnf provided more excellentresults than L-15+5% PEG.

(2) α1-Antitrypsin Synthesis Capacity and CYP3A4 Inducing Capacity

Monkey hepatocytes were maintained at 25° C. in Lnf or in L-15+5% PEGfor 2 days (48 hours) or 4 days (96 hours). subsequently, the cells werecultured in Lnf at 37° C., and α1-antitrypsin synthesis capacity of thecells was measured. The results are shown in FIGS. 2 and 3. The datashown in FIGS. 2 and 3 are relative values with respect to thoseobtained from the test where the cells were stored and cultured at 37°C. from the beginning.

As is apparent from FIGS. 2 and 3, in the case where cells were culturedafter having been stored at 25° C. for 2 or 4 days, as compared with thecase where cells were stored and cultured at 37° C. from the beginning,higher α1-antitrypsin synthesis capacity was maintained through theculture period of 8 to 15 days.

Separately, monkey hepatocytes were maintained in Lnf or L-15+5% PEG at25° C. for 2 days (48 hours). Subsequently, the cells were cultured inLnf at 37° C., and the CYP3A4 inducing capacity of the cells wasmeasured. The results are shown in FIG. 4. FIG. 4 also shows relativevalues obtained from the case where the cells were stored and culturedat 37° C. from the beginning.

As is apparent from FIG. 4, in the case where the cells were culturedafter having been maintained at 25° C. for 2 days, strong CYP3A4inducing capacities (2.3 times and 2.4 times) were observed on day 10 today 14 of culture, as compared with the case where the cells were storedand cultured at 37° C. from the beginning (1.8 times).

(3) Effect on Urea Synthesis Capacity (Human Hepatocytes)

Human hepatocytes were maintained in Lnf or Isom at 25° C. for 2, 4 or 6days. Subsequently, the cells were cultured at 37° C. in Lnf, and theurea synthesis capacity of the cells was measured. The results are shownin FIGS. 5, 6, and 7.

As is apparent from FIGS. 5, 6 and 7, as compared with the controlgroups in which the cells were maintained and cultured at 37° C. fromthe beginning, the cells that were cultured after having been maintainedin Lnf or Isom at 25° C. for 2, 4 or 6 days exhibited strong ureasynthesis capacities.

(4) Albumin Synthesis Capacity and α1-Antitrypsin Synthesis Capacity(Human Hepatocytes)

Human hepatocytes were maintained in Lnf or Isom at 25° C. for 4 days.Subsequently, the cells were cultured at 37° C. in Lnf, and the albuminsynthesis capacity and α1-antitrypsin synthesis capacity were measured.The results are shown in FIGS. 8 and 9.

As is apparent from FIGS. 8 and 9, as compared with the control groupsin which the cells were maintained and cultured at 37° C. from thebeginning, the cells that were cultured after having been maintained at25° C. for 4 days exhibited strong albumin synthesis capacity and strongα1-antitrypsin synthesis capacity.

(5) CYP3A4 Inducing Capacity (Human Hepatocytes)

Human hepatocytes were maintained in Lnf or L-15+5% PEG at 25° C. for 2,4 or 6 days. Subsequently, the cells were cultured at 37° C. in Lnf, andthe CYP3A4 inducing capacity of the cells was measured. The results areshown in FIG. 10.

As is apparent from FIG. 10, as compared with the control groups inwhich the cells were maintained and cultured at 37° C. from thebeginning, the cells that were cultured after having been maintained inLnf or L-15+5% PEG at 25° C. for 2, 4, or 6 days exhibited strong CYP3A4inducing capacity.

INDUSTRIAL APPLICABILITY

The method of the present invention enables long-term culturing ofhepatocytes without permitting reduction in enzymatic activity orenzyme-inducing activity. Moreover, since the cells can be maintained ataround room temperature for 1 to 6 days, the method of the invention isvery useful for a long travel of hepatocytes after isolation.

1. A method of culturing hepatocytes for a long term, characterized inthat fresh hepatocytes are maintained in a medium at 15 to 30° C. for 1to 6 days, followed by culturing under physiological conditions.
 2. Themethod of culturing hepatocytes for a long term according to claim 1,wherein the medium for fresh hepatocytes contains at least Lanford'smedium.
 3. The method of culturing hepatocytes for a long term accordingto claim 1 or 2, wherein the culturing under physiological conditions isperformed at 37±1° C. in a medium containing at least Lanford's medium.